The need for quantitative determination of the concentration of a substance of interest in a test sample is widespread and varied in application. Such need arises prominently in connection with clinical laboratory tests, quality control in the production of any mixture of materials, for example, food, agricultural or other mixtures; and in analytical assessment of, for example, waste products such as industrial or municipal waste. Methods of quantitation are varied but generally involve generation of some detectable material such as a colored substance, a substance absorbing ultraviolet or infrared light at a particular wavelength, a fluorescent material, or, in general, a material which absorbs or generates radiation of particular energy. Such radiation levels can be detected through instrumentation using instruments of complexity ranging from that of a simple colorimeter, or even a color comparator chart, to sophisticated variable wavelength specific infrared spectrometers. Additional detection methods rely on characteristic properties of either the substance to be determined or of a material into which it can be converted, which are assessed by more complex means, as, for example, mass spectroscopy, nuclear magnetic resonance spectroscopy, or stimulated emission of x-radiation.
The expense and complexity of these analytical procedures are, of course, proportional to the sophistication of the instrumentation required (although a highly complex instrument can sometimes eliminate many operator manipulations). Especially for preliminary screening tests, and for tests which are useful to individual consumers, it would be desirable to formulate testing procedures which obviate the need for expensive instrumentation and which are simple to perform. The present invention provides a method for quantitation of desired analytes which eliminates the need for such complication and expense. It provides a testing method which is usable unaided by instrumentally based analog measurements and gives a digital readout useable to virtually anyone.
An attempt has been made to obtain a digital readout in tests for glucose using a series of oxidation reactions containing varying amounts of reducing agent to prevent the formation of an oxidized indicator, as disclosed by Hochstrasser in U.S. Pat. No. 3,964,871. However, this method is limited to analytes which are capable of generating peroxide, and even for these analytes the results cannot be finely tuned to a desired level of quantitation.
The method of the present invention is grounded on the combination and integration of two concepts, the competition for a substrate by each of a pair of catalysts, and an orderly array of varying levels of competition. The competition aspect per se thus differs from that of, for example, antigen for antibody as in radioimmunoassay (RIA) or enzyme labeled immunosorbent assay (ELISA).
Recently, the use of two enzymes simultaneously has been applied to the problems of extending the range of analyte concentration which can be assessed (German patent application Publication No. DE 3211167A1, published Sep. 29, 1983). The catalysts used in this disclosure were employed in such a way that a cofactor for one catalyst having a detectable product was used up substantially below the level represented by the cofactor for the other, so that the analyte range measured by one catalyst was different from that measured by the other. No means for digital readout was provided, and no advantage was taken of competition, as the two catalysts function substantially independently.
The present invention relies on competition for substrate, as the amount of substrate available for a control reaction is dependent on the relative amount of catalyst for a competing reaction present. In addition, by serially varying the relative amount of competing reaction catalyst the amount of substrate remaining can be left at a detectable level or not. As the relative amount of competing catalyst can be determined by the will of the experimenter (or by a series of pre-set levels chosen by the manufacturer), no instrumentation is required for its quantitation.